Isolation and characterization of spontaneous spheroid aggregates within human colon carcinomas

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14515-14515
Author(s):  
V. Dangles-Marie ◽  
P. Validire ◽  
S. Richon ◽  
L. Weiswald ◽  
M. Briffod ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Colon ◽  
Cancer Cell Lines ◽  
Mesenchymal Transition ◽  
Isolation And Characterization

14515 Background: In vitro spheroid model using cancer cell lines is widely admitted to mimic in vivo micro tumors, including micrometastases. Floating spheroid cell cluster culture has been recently used for normal and cancer stem cell expansion. Spontaneously spheroids generated in vivo have been only studied in ovarian cancer ascites while organoid aggregates have been sometimes observed in the establishment of human colon cancer cell lines. In this study, we investigated whether spontaneous spheroid aggregates from colon cancer could be isolated and characterized. Methods: 127 colorectal primary tumor specimens have been collected and mechanically dissociated into small fragments, which were then shortly cultured on cell plastic flask. Production of spheroid- like structures, referred to as colospheres, was examined at Day 1 and colospheres were gathered for phenotypic characterization. Results: Colospheres were successfully generated from 67 surgical specimens (53%). The capacity to form colospheres was strictly restricted to tumor tissue: dissociated normal colon mucosa never generated colospheres and colospheres were formed exclusively by cancer cells. The ability to generate colospheres was demonstrated to be significantly related to tumor aggressiveness, according to nodal status and AJCC’s stages (Chi-2 test, p<0.05). Immunohistochemical studies showed that cells forming colospheres were frequently positive for Ki67, and displayed often a disturbed expression of the epithelial caretaker E-cadherin. Peripheral cells of colospheres were able to migrate into Matrigel in absence of any chemoattractant. Conclusions: Collectively, the morphology of these colospheres derived directly from tumoral tissues and made up exclusively of cancer cells, their potential capacity to acquire an epithelial-to-mesenchymal transition phenotype and their in vitro migration ability could be aligned with the collective migration properties of carcinomas. Consequently, these ex vivo spherical structures might form an in vitro cell system for micrometastasis studies, at the very time when mortality among colorectal cancer patients continues to be attributed to metastasis development. No significant financial relationships to disclose.

Avenanthramides Inhibit Proliferation of Human Colon Cancer Cell Lines In Vitro

2010 ◽  
Vol 62 (8) ◽  
pp. 1007-1016 ◽  
Author(s):  
Weimin Guo ◽  
Lin Nie ◽  
Dayong Wu ◽  
Mitchell L. Wise ◽  
F. William Collins ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Colon ◽  
Cancer Cell Lines ◽  
Colon Cancer Cell ◽  
Human Colon Cancer Cell ◽  
Human Colon Cancer ◽  
Colon Cancer Cell Lines

scholarly journals NF-κB maintains the stemness of colon cancer cells by downregulating miR-195-5p/497–5p and upregulating MCM2

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Longgang Wang ◽  
Jinxiang Guo ◽  
Jin Zhou ◽  
Dongyang Wang ◽  
Xiuwen Kang ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Human Colon ◽  
Colon Cancer Cells ◽  
Tumor Models ◽  
Human Colon Cancer ◽  
Subcutaneous Tumor

Abstract Background Colon cancer represents one of the leading causes of gastrointestinal tumors in industrialized countries, and its incidence appears to be increasing at an alarming rate. Accumulating evidence has unveiled the contributory roles of cancer stem cells (CSCs) in tumorigenicity, recurrence, and metastases. The functions of NF-kappa B (NF-κB) activation on cancer cell survival, including colon cancer cells have encouraged us to study the role of NF-κB in the maintenance of CSCs in colon cancer. Methods Tumor samples and matched normal samples were obtained from 35 colon cancer cases. CSCs were isolated from human colon cancer cell lines, where the stemness of the cells was evaluated by cell viability, colony-forming, spheroid-forming, invasion, migration, and apoptosis assays. NF-κB activation was then performed in subcutaneous tumor models of CSCs by injecting lipopolysaccharides (LPS) i.p. Results We found that NF-κB activation could reduce the expression of miR-195-5p and miR-497-5p, where these two miRNAs were determined to be downregulated in colon cancer tissues, cultured colon CSCs, and LPS-injected subcutaneous tumor models. Elevation of miR-195-5p and miR-497-5p levels by their specific mimic could ablate the effects of NF-κB on the stemness of colon cancer cells in vivo and in vitro, suggesting that NF-κB could maintain the stemness of colon cancer cells by downregulating miR-195-5p/497–5p. MCM2 was validated as the target gene of miR-195-5p and miR-497-5p in cultured colon CSCs. Overexpression of MCM2 was shown to restore the stemness of colon cancer cells in the presence of miR-195-5p and miR-497-5p, suggesting that miR-195-5p and miR-497-5p could impair the stemness of colon cancer cells by targeting MCM2 in vivo and in vitro. Conclusions Our work demonstrates that the restoration of miR-195-5p and miR-497-5p may be a therapeutic strategy for colon cancer treatment in relation to NF-κB activation.

Incomplete processing of progastrin expressed by human colon cancer cells: role of noncarboxyamidated gastrins

1994 ◽  
Vol 266 (3) ◽  
pp. G459-G468 ◽  
Author(s):  
P. Singh ◽  
Z. Xu ◽  
B. Dai ◽  
S. Rajaraman ◽  
N. Rubin ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Colon ◽  
Cancer Cell Lines ◽  
Colon Cancer Cell ◽  
Colon Cancer Cells ◽  
Human Colon Cancer ◽  
Colon Cancer Cell Lines

Gastrin is mitogenic for several colon cancers. To assess a possible autocrine role of gastrin in colon cancers, we examined human colon cancer cell lines for expression of gastrin mRNA and various forms of gastrin. Gastrin mRNA was not detected in the majority of colon cancer cell lines by Northern hybridization but was detected in all human colon cancer lines by the sensitive method of reverse transcriptase-polymerase chain reaction (PCR). Gastrin mRNA was quantitated by the competitive PCR method. The majority of cell lines expressed very low levels of gastrin mRNA (< 1-5 copies/cell); only one cell line expressed > 20 copies/cell. The mature carboxyamidated form of gastrin was not detected in any of the cell lines by radioimmunoassay or immunocytochemistry. Results suggested that either gastrin mRNA expressed by colon cancer cells was altered (mutated) or posttranslational processing of progastrin was incomplete. Gastrin cDNA from all the colon cancer cell lines had an identical sequence to the published sequence of human gastrin cDNA. Specific antibodies against precursor forms of gastrin were used, and significant concentrations of nonamidated (glycine-extended) and prepro forms of gastrin were measured in tumor extracts of representative colon cancer cell lines. The presence of precursor forms of gastrin suggested a lack of one or more of the processing enzymes and/or cofactors. Significant concentrations of the processing enzyme (peptidylglycine alpha-amidating monooxygenase) were detected in colon cancer cells by immunocytochemistry. Therefore, lack of other cofactors or enzymes may be contributing to incomplete processing of precursor forms of gastrin, which merits further investigation. Since low levels of gastrin mRNA were expressed by the majority of human colon cancer cell lines and progastrin was incompletely processed, it seems unlikely that gastrin can function as a viable autocrine growth factor for colon cancer cells. High concentrations of glycine-extended gastrin-17 (GG) (> 10(-6) M) were mitogenic for a gastrin-responsive human colon cancer (DLD-1) cell line in vitro. It remains to be seen if GG or other precursor forms of gastrin are similarly mitogenic in vivo, which may then lend credibility to a possible autocrine role of gastrinlike peptides in colon cancers.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Combination of oxaliplatin and irinotecan on human colon cancer cell lines: activity in vitro and in vivo

2001 ◽  
Vol 12 (9) ◽  
pp. 741-751 ◽  
Author(s):  
Sylvie Guichard ◽  
Stéphanie Arnould ◽  
Isabelle Hennebelle ◽  
Roland Bugat ◽  
Pierre Canal
Keyword(s):  
Colon Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Colon ◽  
Colon Cancer Cell ◽  
Human Colon Cancer Cell ◽  
Human Colon Cancer ◽  
Colon Cancer Cell Lines

scholarly journals 725 Immunomodulation by targeting PDL-1 in colon cancer using nuclear receptor 4A1 (NR4A1) antagonists

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A754-A755
Author(s):  
Maen Abdelrahim ◽  
Kumaravel Mohankumar ◽  
Keshav Karki ◽  
Stephen Safe
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Colon Cancer Cell ◽  
Colon Cancer Cells ◽  
Western Blots ◽  
Colon Cancer Cell Lines

BackgroundThe nuclear orphan receptor 4A1 (NR4A1, Nur77, TR3) is overexpressed in multiple solid tumors including colorectal tumors and is a negative prognostic factor for patient survival.1–3 NR4A1 is expressed in colon cancer cells and exhibit pro-oncogenic activity4 and results of examination of several colon cancer cell lines show that PD-L1 expression is limited and NR4A1 and PD-L1 are co-expressed in SW480 and RKO colon cancer cell lines. Previous studies showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter.5–7 Knockdown of NR4A1 or Sp1 by RNA interference or treatment with mithramycin an inhibitor of Sp-mediated transcription decreased expression of PD-L1 in RKO and SW480 colon cancer cell lines.MethodsSW480, RKO and MC-38 cells were used in this study. Cells were treated for 24 hrs with DIM series of compounds.ResultsCurrent data coupled with ongoing gene expression and PD-L1 promoter studies demonstrate that PD-L1 expression is regulated by NR4A1/Sp1 in colon cancer cells (figures 1–3). Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways. Treatment of RKO and SW480 colon cancer cell lines with a series of potent 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl) analogs decreased expression of PD-L1. These results show that bis-indole derived NR4A1 antagonists act as small molecule mimics of immunotherapeutics that target PD-L1. In vivo applications of NR4A1 ligands that target PD-L1 and their effects on tumor growth and immune surveillance are currently being investigated.ConclusionsBis-indole derived NR4A1 antagonists inhibit PD-L1 expression. NR4A1/SP1 regulates PD-L1 and is inhibited by NR4A1 antagonist. NR4A1 ligands such as DIM-3-Br-5-OCF3 were among the most potent of the substituted DIM compounds and ongoing in vivo studies show that this DIM compound also inhibits tumor growth in a syngenic mouse model (data not shown). Data from this study demonstrate the pro-oncogenic activity of NR4A1 and show that the synthetic buttressed analog DIM-3-Br-5-OCF3 acts as an NR4A1 antagonist and inhibits PD-L1 expression. These drugs can be developed for future clinical applications.Referenceswww.cancer.org/cancer/colon-rectal-cancer/about/key-statistics.html.Garcia-Villatoro et al., Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis. Am J Physiol Gastrointest Liver Physiol 2020;318(3):G451–G463.Safe S, Jin UH, Hedrick E, et al. Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 2014;28(2):157–72.Maxwell MA, Muscat GE. The NR4A subgroup: immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal 2006;4:e002.Lee SO, Li X, Hedrick E, et al. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells. Mol Endocrinol 2014;28(10):1729–39.Safe S, Kim K. Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 2008;41(5):263–75.Tao LH, Zhou XR, Li FC, Chen Q, Meng FY, Mao Y, et al. A polymorphism in the promoter region of PD-L1 serves as a binding-site for SP1 and is associated with PD-L1 overexpression and increased occurrence of gastric cancer. Cancer Immunol Immunother 2017;66(3):309–18.Abstract 725 Figure 1NR4A1 inactivation inhibits PD-L1 expression. SW480, RKO and MC-38 cells were transfected with siCtrl (non-specific oligonucleotide) and two oligonucleotides targeting NR4A1 (siNR4A1(1) and siNR4A1(2)) or PD-L1 (siPD-L1(1) and siPD-L1(2)) for 72 hrss. Protein expression from whole cell lysates were analyzed by western blots and effects on PD-L1 expression were determinedAbstract 725 Figure 2Sp1 inactivation inhibits PD-L1 expression. SW480, RKO and MC-38 cells were transfected with siCtrl and oligonucleotides targeting Sp1 (siSp1(1) and siSp1(2)) for 72 hrs as well as treated with Mithrsamycin (150 and 300 nM) for 24 hrs. Protein expression from was analyzed by western blots and effects on PD-L1 levels were determined.Abstract 725 Figure 3Role of NR4A1/Sp in regulation of PD-L1. SW480, RKO and MC-38 cells were treated with DIM-3-Br-5-OCF3 for 24 hrss and protein interactions with the GC-rich PD-L1 promoter region were analyzed by ChIP using primers encompassing GC-rich region of the promoter

scholarly journals Met-HER3 crosstalk supports proliferation via MPZL3 in MET-amplified cancer cells

2021 ◽  
Author(s):  
Yaakov Elisha Stern ◽  
Stephanie Duhamel ◽  
Abdulhameed Al-Ghabkari ◽  
Anie Monast ◽  
Benoit Fiset ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Tyrosine Kinases ◽  
Human Cancer ◽  
Transmembrane Protein ◽  
Cancer Cell Lines ◽  
Her Family

Receptor tyrosine kinases (RTKs) are recognized as targets of precision medicine in human cancer upon their gene amplification or constitutive activation, resulting in increased downstream signal complexity including heterotypic crosstalk with other RTKs. The Met RTK exhibits such reciprocal crosstalk with several members of the human EGFR (HER) family of RTKs when amplified in cancer cells. We show that Met signaling converges on HER3 tyrosine phosphorylation across a panel of seven MET-amplified cancer cell lines and that HER3 is required for cancer cell expansion and oncogenic capacity in-vitro and in-vivo. Gene expression analysis of HER3-depleted cells identified MPZL3, encoding a single-pass transmembrane protein, as a HER3-dependent effector in multiple MET-amplified cancer cell lines. MPZL3 interacts with HER3 and MPZL3 loss phenocopies HER3 loss in MET-amplified cells, while MPZL3 overexpression rescues proliferation upon HER3 depletion. Together, these data support an oncogenic role for a HER3-MPZL3 axis in MET-amplified cancers.

Sign in / Sign up

Export Citation Format

Share Document